A number of tubulin domains have been studied by a variety of methods. (1) We have found that benzophenanthridines constitute a new class of antimicrotubule agents. Chelidonine interacts at the colchicine site whereas sanguinarine and chelerythrine interact with tubulin through reversible pseudobase formation by nucleophilic attack. (2) Colchicine binds as the interface between alpha and beta tubulin with primary binding on beta and secondary binding on alpha. There are three alpha-domains involved in binding and indirect evidence suggests that it is the B ring of colchicine that is in contact with alpha tubulin. (3) Colchicine binding leads to long range conformational effects on beta-tubulin that lead to the unfolding of the C-terminus which, in turn, prevents addition of new dimers during polymerization and may explain the coupling of hydrolysis of the GTP cap to polymerization. (4) Very low (<1M) concentration of urea modify numerous properties of tubulin in the order decreased polymerization > decreased GTPase > enhanced fluorescence of a colchicine analogue > decreased proteolytic accessibility > enhanced ANS fluorescence. These can be shown to be independent regions and suggest multistep unfolding of the protein. Physical correlates are now being sought.